FIGURE 5.

LOS-IV biosynthesis mutant shows increased virulence in zebrafish embryo infection model. Infection of zebrafish embryos with red fluorescent wild-type M. marinum E11 (WT) and mutants A3 and A8, A3 complemented with pUC-Int-cat-mmar_2319-22 (A3c) and ESX-1 mutant eccCb1::tn (ESX1), were monitored at 5 days post-infection. A, representative overlay pictures of embryos infected with WT, A3, A3c, and A8 with ∼80 colony-forming units (CFU). B, automated quantification of infection based on red pixel intensity (31). Represented are relative infection levels compared with wild-type infection as a log of the percentage. Results of three replicate experiments, with the average injected cfu below each group, are shown with standard deviation (***, p < 0.001 by unpaired Student's t test). C, mean bacterial loads, measured as cfu, for zebrafish embryos infected for 5 days with WT and mutant A8 bacteria. Error bars represent mean ± S.E. D, growth curve of bacterial cultures in 7H9 + 0.05% Tween 80; mean A₆₀₀ measurements of three independent cultures with standard deviation.